Orexin signaling is mediated by two receptors and two peptide agonists. The peptides (orexin A and orexin B) are cleavage products of the same gene, pre-pro orexin. In the central nervous system, neurons producing pre-pro orexin are found in the perifornical nucleus, the dorsal hypothalamus and the lateral hypothalamus (Peyron et al., 1998, J. Neurosci. 18: 9996-10015). Orexigenic cells in these regions project to many areas of the brain, extending rostrally to the olfactory bulbs and caudally to the spinal cord (Van den Pol, 1999, J. Neurosci. 19: 3171-3182). The orexins bind to two high affinty receptors, referred to as orexin-1 and orexin-2 receptors. The orexin-1 receptor is selective in favor of orexin A, while the orexin-2 receptor binds both orexins with similar affinities.
The broad CNS distribution of cells producing orexin, as well as cells expressing the orexin receptors, suggests involvement of orexin in a number of physiological functions, including feeding, drinking, arousal, stress, metabolism and reproduction. A recent report describing targeted necrosis of cells producing pre-pro orexin suggests that the most physiologically important roles of the orexins may be effects on arousal, feeding and metabolism (Hara et al., 2001, Neuron 30: 345-354).
Several lines of evidence indicate that the orexin system is an important modulator of arousal. Rodents administered orexin intracerebroventricularly spend more time awake (Piper et al., 2000, J. Neurosci. 12: 726-730. Orexin-mediated effects on arousal have been linked to orexin neuronal projections to histaminergic neurons in the tuberomammillary nucleus (TMN) (Yamanaka et al., 2002, Biochem. Biophys. Res. Comm. 290: 1237-1245). TMN neurons express the orexin-2 receptor primarily, and the orexin-1 receptor to a lesser extent. Rodents whose pre-pro orexin gene has been knocked out, or whose orexigenic neurons have been killed, display altered sleep/wake cycles similar to narcolepsy (Chemelli et al., 1999, Cell 98: 437-451; Hara et al., 2001, supra). Dog models of narcolepsy have been shown to have mutant or non-functional orexin-2 receptors (Lin et al., 1999, Cell 98: 365-376). Human narcolepsy appears to be linked to deficient orexin signaling, likely related to immune ablation of orexinergic neurons in the lateral hypothalamus (Mignot et al., 2001, Am. J. Hum. Genet. 68: 686-699; Minot & Thorsby, 2001, New England J. Med. 344: 692), or, in rare cases, to mutations in the orexin-2 gene (Peyron et al., 2000, Nature Med. 6: 991-997).
Disorders of the sleep-wake cycle are therefore likely targets for orexin-2 receptor modulator activity. Examples of sleep-wake disorders that may be treated by agonists or other modulators that up-regulate orexin-2 receptor-mediated processes include narcolepsy, jet lag (sleepiness) and sleep disorders secondary to neurological disorders such as depression. Examples of disorders that may be treated by antagonists or other modulators that down-regulate orexin-2 receptor-mediated processes include insomnia, restless leg syndrome, jet lag (wakefulness) and sleep disorders secondary to neurological disorders such as mania, schizophrenia, pain syndromes and the like.
The orexin system also interacts with brain dopamine systems. Intracerebroventricular injections of orexin in mice increase locomotor activity, grooming and stereotypy, these behavioral effects are reversed by administration of D2 dopamine receptor antagonists (Nakamura et al., 2000, Brain Res. 873: 181-187). Therefore, orexin-2 modulators may be useful to treat various neurological disorders; e.g., agonists or up-regulators to treat catatonia, antagonists or down-regulators to treat Parkinson's disease, Tourette's syndrome, anxiety, delerium and dementias.
Orexins and their receptors have been found in both the myenteric and submucosal plexus of the enteric nervous system, where orexins have been shown to increase motility in vitro (Kirchgessner & Liu, 1999, Neuron 24: 941-951) and to stimulate gastric acid secretion in vitro (Takahashi et al., 1999, Biochem. Biophys. Res. Comm. 254: 623-627). Orexin effects on the gut may be driven by a projection via the vagus nerve (van den Pol, 1999, supra), as vagotomy or atropine prevent the effect of an intracerebroventricular injection of orexin on gastric acid secretion (Takahashi et al., 1999, supra). Orexin receptor antagonists or other down-regulators of orexin receptor-mediated systems are therefore potential treatments for ulcers, irritable bowel syndrome, diarrhea and gastroesophageal reflux.
Body weight may also be affected by orexin-mediated regulation of appetite and metabolism. Some effects of orexin on metabolism and appetite may be mediated in the gut, where, as mentioned, orexins alter gastric motility and gastric acid secretion. Orexin antagonists therefore are likely to be useful in treatment of overweight or obesity and conditions related to overweight or obesity, such as insulin resistance/type II diabetes, hyperlipidemia, gallstones, angina, hypertension, breathlessness, tachycardia, infertility, sleep apnea, back and joint pain, varicose veins and osteoarthritis. Conversely, orexin agonists are likely to be useful in treatment of underweight and related conditions such as hypotension, bradycardia, ammenorrhea and related infertility, and eating disorders such as anorexia and bulimia.
Intracerebroventricularly administered orexins have been shown to increase mean arterial pressure and a heart rate in freely moving (awake) animals (Samson et al., 1999, Brain Res. 831: 248-253; Shirasaka et al., 1999, Am. J. Physiol. 277: R1780-R1785) and in urethane-anesthetized animals (Chen et al., 2000, Am. J. Physiol. 278: R692-R697), with similar results. Orexin receptor agonists may therefore be candidates for treatment of hypotension, bradycardia and heart failure related thereto, while orexin receptor antagonists may be useful for treatment of hypertension, tachycardia and other arrhythmias, angina pectoris and acute heart failure.
From the foregoing discussion, it can be seen that the identification of orexin receptor modulators, particularly modulators of the orexin-2 receptor, will be of great advantage in the development of therapeutic agents for the treatment of a wide variety of disorders that are mediated through these receptor systems. There exists a need in the art for improved methods for identifying modulators of human orexin-2 receptor, particularly methods that do not require the use of recombinant DNA molecules encoding the human orexin-2 receptor. Such improved methods can facilitate the rapid processing of chemical libraries to identify modulators of the human orexin-2 receptor, and preferably will also be amenable to automation, thereby providing substantial commercial advantages for new drug discovery and development applications. The present invention is believed to satisfy these needs and to provide other related advantages.
Citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention. All publications referred to herein are incorporated by reference in their entireties.